Upregulation of Metastasis-Associated Gene 2 Promotes Cell Proliferation and Invasion in Nasopharyngeal Carcinoma

Journal name: OncoTargets and Therapy Article Designation: Original Research Year: 2016 Volume: 9 OncoTargets and Therapy Dovepress Running head verso: Wu et al Running head recto: MTA2 promotes proliferation and invasion of NPC cell open access to scientific and medical research DOI: http://dx.doi.org/10.2147/OTT.S96518

Open Access Full Text Article Original Research Upregulation of metastasis-associated gene 2 promotes cell proliferation and invasion in nasopharyngeal carcinoma

Minhua Wu1,2,* Aims: Metastasis-associated gene 2 (MTA2) is reported to play an important role in tumor Xiaoxia Ye2,* progression, but little is known about the role of MTA2 in nasopharyngeal carcinoma (NPC). Xubin Deng3,* The aim of the study was to explore the expression and function of MTA2 in NPC. Yanxia Wu4 Methods: Expression of MTA2 in NPC tissues and cell lines was detected by immunohistochem- Xiaofang Li4 istry and Western blotting. Relationship between MTA2 expression and clinicopathological Lin Zhang1 features was analyzed. Stable MTA2-overexpressing and MTA2-siliencing NPC cells were established by transfection with plasmids encoding MTA2 cDNA and lentivirus-mediated short 1 Department of Histology and hairpin RNA, respectively. Cell viability was determined by Cell Counting Kit-8 and colony Embryology, Southern Medical University, Guangzhou, 2Department formation assay. Cell migration ability was evaluated by wound healing and transwell invasion

For personal use only. of Histology and Embryology, assay. The impact of MTA2 knockdown on growth and metastasis of CNE2 cells in vivo was Guangdong Medical University, determined by nude mouse xenograft models. Expression of several Akt pathway proteins was Zhanjiang, 3Affiliated Cancer Hospital of Guangzhou Medical University, detected by Western blotting. Cancer Center of Guangzhou Medical Results: MTA2 was upregulated in NPC tissues and three NPC cell lines detected (CNE1, University, Guangzhou, 4Pathological Diagnosis and Research Center, CNE2, and HNE1). MTA2 expression was related to clinical stage and lymph node metastasis Affiliated Hospital of Guangdong of patients with NPC. MTA2 upregulation promoted proliferation and invasion of CNE1 cells, Medical University, Zhanjiang, while MTA2 depletion had opposite effects on CNE2 cells. Moreover, MTA2 depletion sup- People’s Republic of China pressed growth and metastasis of CNE2 cells in vivo. MTA2 overexpression activated Akt and *These authors contributed equally upregulated the expression of matrix metalloproteinase 7 and cyclin D1. to this work Conclusion: We conclude that MTA2 acts as an oncogene in tumorigenesis of NPC. MTA2 may be a potential target for gene therapy in NPC. OncoTargets and Therapy downloaded from https://www.dovepress.com/ by 54.70.40.11 on 13-Dec-2018 Keywords: MTA2, nasopharyngeal carcinoma, proliferation, invasion, Akt pathway

Introduction Nasopharyngeal carcinoma (NPC) is one of the most prevalent cancers in Southeast Asia and South China. Although the treatment of NPC has improved greatly in recent years with the development of radiotherapy, the 5-year survival rate of patients with NPC is still ,60%. Approximately 20,000 people die of NPC every year in the People’s Republic of China.1,2 The main cause of treatment failure and patient’s mortality is tumor metastasis, but the molecular mechanism of tumorigenesis and metastasis of

Correspondence: Lin Zhang NPC are still elusive. It will be of great clinical value to find out new biomarkers Department of Histology and involved in the progression of NPC. Embryology, Southern Medical University, Metastasis-associated gene (MTA) family is a novel gene family.3 MTA1, MTA2, 1838 North Guangzhou Avenue, Guangzhou 510515, People’s Republic and MTA3 are the main members of this family. They are components of nucleosome of China remodeling and deacetylation complex.4 As a part of the nucleosome remodeling and Tel +86 20 164 8205 Email [email protected] deacetylation complex, MTAs are proved to modulate gene transcription by affecting

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chromatin remodeling.5,6 MTA2, as a member of MTA gene were blinded to the patient’s clinicopathological information. family, is highly expressed in human osteosarcoma,7 colorec- The degree of immunohistochemical staining was assessed tal cancer,8 ovarian cancer,9 lung cancer,10 and several other by the positive cell proportion and intensity of staining.18 The human tumor tissues. High MTA2 expression is positively percentage of positivity was scored as follows: 0 (,5%), related to metastasis and poor prognosis in esophageal 1 (5%–25%), 2 (26%–50%), and 3 (>50%). The staining carcinoma11 and pancreatic carcinoma.12 intensity was scored as follows: 0 (no), 1 (light yellow), Expression of MTA2 affects tumor cell growth, metas- 2 (yellow), and 3 (brown yellow). The final score was calcu- tasis, apoptosis, and cell skeleton.13,14 Silencing of MTA2 lated by multiplying the proportion score and intensity score. reverses the malignant phenotypes of glioma,15 gastric Slides with a final score of $4 were defined as positive; cancer,16 and breast cancer17 cells. These studies strongly otherwise, they were defined as negative. suggest a correlation between tumor development and MTA2 expression. However, to the best of our knowledge, Cell culture the expression pattern and function of MTA2 in NPC have Human NPC cell lines CNE1, CNE2, and HNE1 and human not been reported before. immortalized nasopharyngeal epithelium cell line NP6919 were obtained from the Cancer Research Institute of Guangdong Materials and methods Medical University and Southern Medical University, Guang- Patients and samples dong, People’s Republic of China. The use of these cell lines Samples of 107 NPC tissues and 28 noncancerous nasopha- were approved by the institutional review boards of both of the ryngeal epithelium tissues were obtained from biopsy aforementioned schools. CNE1, CNE2, and HNE1 cells were of patients from the Affiliated Hospital of Guangdong cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) Medical University (Zhanjiang, People’s Republic of China) supplemented with 10% fetal bovine serum (FBS; Thermo

between 2010 and 2011. None of the patients with NPC Fisher Scientific, Waltham, MA, USA) at 37°C with 5% CO2 received radiotherapy or chemotherapy before biopsy. The and a humidified atmosphere. NP69 cells were grown in kera- For personal use only. clinicopathological information was obtained from medical tinocyte serum-free medium (Thermo Fisher Scientific). records. Staging of cancer was in accordance with the seventh American Joint Committee on Cancer and the International Quantitative reverse transcription– Union for Cancer Control cancer staging guidelines issued polymerase chain reaction analysis in 2010. The study was approved by the Medical Ethics Quantitative real-time reverse transcription–polymerase Committee of the Affiliated Hospital of Guangdong Medical chain reaction (qRT-PCR) assay was performed as previously University. Written informed consent was obtained from each described.20 Briefly, RNAiso Plus reagent (Takara, Dalian, patient. Patients consisted of 71 males and 36 females with People’s Republic of China) was used to extract total RNA of an average age of 48 years (range: 18–75 years). cells. The cDNA was obtained from messenger RNA with a Primescript™ RT reagent kit (Takara). A SYBR Green PCR OncoTargets and Therapy downloaded from https://www.dovepress.com/ by 54.70.40.11 on 13-Dec-2018 Immunohistochemistry kit (Takara) was used in the amplification processes by a Light Sections from patients were deparaffinized with xylene Cycler system (Hoffman-La Roche Ltd, Basel, Switzerland). and rehydrated with graded alcohol. For antigen retrieval, The primers used for amplification of MTA2 andβ -actin were sections were immersed in citrate buffer (pH =6.0) and as follows: sense 5′-GATGAGATGGAGGAATGGTCAG-3′, microwaved for 5 minutes. To eliminate intrinsic peroxidase antisense 5′-ACCTGTTTCAGTTTGCTGTCTG-3′ (for

activity, slides were incubated with 3% H2O2 for 8 minutes. MTA2); sense 5′-TGACGTGGACATCCGCAAAG-3′, Slides were incubated overnight at 4°C with a goat antibody and antisense 5′-CTGGAAGGTGGACAGCGAGG-3′ (for against MTA2 (dilution, 1:300; Santa Cruz Biotechnology β-actin). The cycling program was as follows: denaturation Inc., Dallas, TX, USA). After washing with phosphate- at 95°C for 30 seconds, followed by 40 cycles of denaturing buffered saline (PBS), sections were detected with a Polink-2 at 95°C for 5 seconds, annealing at 60°C for 30 seconds. Plus Polymer HRP Kit (GBI, Mukilteo, WA, USA) according The qRT-PCR result was analyzed with the 2−ΔΔCt method.21 to the manufacturer’s instructions. Signals were visualized β-actin was used as an internal control. with 3,3′-diaminobenzidine tetrahydrochloride for 2 minutes and slightly counterstained with hematoxylin. The primary Western blot analysis antibody was replaced with PBS as a negative control. The Western blot experiment was performed as previously slides were evaluated independently by two investigators who described.22 Briefly, cells or tumor tissues were lysed with RIPA

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Dovepress MTA2 promotes proliferation and invasion of NPC cell

reagent (Beyotime, Shanghai, People’s Republic of China). by qRT-PCR and Western blot. The stable MTA2-silencing The concentration of protein was determined by bicinchoninic and negative control CNE2 cells were named as CNE2/ acid method. An equal amount of protein in each lane was shMTA2 and CNE2/shNC, respectively. subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride mem- CCK-8 proliferation assay branes (EMD Millipore, Billerica, MA, USA). After blocking Cell Counting Kit-8 (CCK-8) proliferation assay was per- for 1 hour with 5% nonfat milk, the polyvinylidene fluoride formed as previously described.23 Briefly, cells were seeded membranes were incubated with MTA2 and β-actin polyclonal in 96-well plates (2,000 cells per well) and grown in com- antibody (dilution, 1:1,000; Santa Cruz Biotechnology Inc.), plete medium for 1 day, 2 days, 3 days, 4 days, or 5 days in

Akt and pAkt (Ser473) monoclonal antibody (dilution, 1:2,000; a humidified atmosphere at 37°C with 5% CO2. For analysis, Cell Signaling Technology, USA), cyclin D1 monoclonal anti- 20 μL of CCK-8 substrate was added to each well. After body (dilution, 1:1,500; Cell Signaling Technology), hypoxia- culturing for 2 hours, the absorbance of wells was measured inducible factor 1α monoclonal antibody (dilution, 1:300; with a microplate reader at 450 nm. Boster Biotechnology, Wuhan, People’s Republic of China), and matrix metalloproteinase 7 (MMP7) polyclonal antibody Colony formation assay (dilution, 1:1,000; Abcam, Cambridge, UK). After incubation Cells were seeded at a density of 100 cells per well in six-well with primary antibody at 4°C overnight, the membranes were plates. After culturing for 12 days in an incubator, the cells were incubated with secondary antibody (dilution, 1:5,000; Santa fixed with methanol and stained with crystal violet. The number Cruz Biotechnology Inc.) at room temperature for 1 hour. of colonies was counted under an inverse microscope. Signals were visualized with the enhanced chemiluminescence detection system (Pierce, Rockford, IL, USA). The intensity Wound healing assay of protein bands was quantified using Quantity One software Cells were seeded with the same number in six-well plates (Bio-Rad Laboratories Inc., Hercules, CA, USA). The protein in complete medium. When cells grew to ~95% confluence, For personal use only. levels were normalized to those of β-actin. scratch wounds were made with 100 μL sterile pipette tips. To remove the suspended cells, the plates were washed with Establishment of stable MTA2- PBS twice. Cells were grown in DMEM containing 1% FBS, overexpressing CNE1 cell and photos were taken in three defined fields at 0 hour and The pcDNA3.1 plasmid encoding full-length MTA2 48 hours, respectively. The closure area of wounds was mea- cDNA sequence and empty vector were purchased from sured with ImageJ software (National Institutes of Health, Biogot Biotechnology (Nanjing, People’s Republic of Bethesda, MD, USA). China). Plasmids were transfected into CNE1 cell by Lipofectamine 2000 (Thermo Fisher Scientific). Stable Transwell invasion assay MTA2-overexpressing CNE1 cell (CNE1/MTA2) and Transwell cell culture inserts (pore size, 8 m; Corning OncoTargets and Therapy downloaded from https://www.dovepress.com/ by 54.70.40.11 on 13-Dec-2018 μ negative control CNE1 cell (vector) were established under Incorporated, Corning, NY, USA) were coated with Matrigel G418 (Sigma-Aldrich Co., St Louis, MO, USA) selection. (BD Biosciences, San Jose, CA, USA). Fifty thousand cells in The efficiency of MTA2 gene transfection was verified by 100 μL serum-free DMEM were seeded in the upper chamber qRT-PCR and Western blot. of the inserts. The bottom chamber was added with complete medium (DMEM containing 10% FBS) as a chemoattractant. Establishment of stable MTA2-silencing After incubation for 18 hours, cells in the upper surface of CNE2 cell the membranes were removed with a cotton swab. Cells that The expression of MTA2 in CNE2 cell was silenced by had invaded to the lower surface of the membranes were lentivirus-mediated short hairpin RNA (shRNA). The len- fixed with methanol for 10 minutes and stained with 0.1% tivirus particles containing shRNA targeting MTA2 and crystal violet for 30 minutes. Cells were observed under a negative control shRNA were purchased from Genechem microscope and counted in five predetermined fields. (Shanghai, People’s Republic of China). For lentivirus infection, CNE2 cells were incubated with lentivirus and In vivo growth and metastasis 5 μg/mL polybrene for 24 hours. After infection, cells were experiments selected by flow cytometry with a green fluorescent protein Four-week-old male BALB/c nu/nu mice were purchased marker. The efficiency of MTA2 knockdown was evaluated from the Guangdong Medical Laboratory Animal Center and

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were bred in a specific pathogen-free condition. All animal analysis of quantitative data. P,0.05 was considered to be study procedures were performed according to institutional statistically significant. guidelines. For in vivo tumor growth experiment, the mice were Results injected subcutaneously in the right subaxillary with 100 μL MTA2 is upregulated in NPC and related cell suspensions (1×106 cells) of control or CNE2/shMTA2 to clinical features of patients cells (n=6 per group). Diameters of tumors were measured We first investigated the expression of MTA2 in NPC with a caliper weekly. The tumor volumes (V ) were calcu- tissues and cell lines. MTA2 protein was higher in NPC lated by the following formula: V = a × b2/2 (a and b repre- tissues (60/107, 56.1%) compared with noncancerous sent the long and short axes of tumor, respectively).24 The nasopharyngeal epithelium tissues (8/28, 28.6%), based on mice were sacrificed after 3 weeks. Tumors were carefully immunohistochemistry analysis. MTA2 protein is predomi- removed and weighed. nantly expressed in the nucleus of tumor cells (Figure 1A). For in vivo metastasis experiment, the mice were injected Compared with nasopharyngeal epithelium cell line NP69, with 100 μL cell suspensions (1×106 cells) of control or MTA2 protein levels were higher in NPC cell lines CNE1, CNE2/shMTA2 cells through the lateral tail vein (n=6 per CNE2, and HNE1 (Figure 1B). We further investigated the group). After 6 weeks, the mice were sacrificed, and the lungs relationship between MTA2 expression and patient’s clini- of mice were removed and subjected to hematoxylin and copathological features. High MTA2 expression was related eosin staining. The number of pulmonary metastasis foci in to more advanced clinical stage and presence of lymph node each mouse was calculated as previously described.25 metastasis but had no relation with patient’s age, sex, and distant metastasis of tumor (Table 1). Statistical analysis Results are presented as mean ± standard deviation of three MTA2 overexpression enhances independent experiments and analyzed by SPSS 13.0 soft- proliferation and migration of CNE1 cell For personal use only. ware (SPSS Inc., Chicago, IL, USA). Chi-square test was in vitro performed for analysis of qualitative data. Student’s t-test Among the three NPC cell lines detected, CNE1 cell had or one-way analysis of variance with least significant dif- the lowest level of MTA2 protein and CNE2 cell had the ference test for multiple comparisons was performed for highest level of MTA2 protein (Figure 1B). For this reason,

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Figure 1 MTA2 is upregulated in NPC. Notes: (A) Representative negative immunostaining in a noncancerous nasopharyngeal epithelium (NT) tissue and positive nuclear immunostaining in a NPC tissue Magnification: ×400. MTA2 expression is higher in NPC tissues compared with noncancerous tissues. (B) MTA2 protein expression in CNE1, CNE2, HNE1 NPC cell lines and NT NP69 cells detected by Western blot (**P,0.01 vs NP69, *P,0.05 vs NP69). Abbreviations: MTA2, metastasis-associated gene 2; NPC, nasopharyngeal carcinoma.

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Dovepress MTA2 promotes proliferation and invasion of NPC cell

Table 1 Relationship between MTA2 expression and clinico MTA2 activates the Akt signaling pathway pathological features of patients with NPC in NPC Variables n MTA2 expression χ2 P-value We investigated whether the Akt signaling pathway was − (%) + (%) involved in the function of MTA2. We detected the pro- Sex tein levels of total Akt, activated Akt (pAkt), and some Male 71 34 (47.9) 37 (52.1) 1.345 0.246 downstream genes closely related to cell growth and inva- Female 36 13 (36.1) 23 (63.9) Age (years) sion. Compared with control cells, MTA2 overexpression ,50 60 29 (48.3) 31 (51.7) 1.078 0.299 induced upregulation of pAkt and cyclin D1 and MMP7 in $50 47 18 (38.3) 29 (61.7) CNE1/MTA2 cell (Figure 4A), while MTA2 knockdown Clinical stage caused downregulation of these genes in CNE2/shMTA2 Stages I–II 18 13 (72.2) 5 (27.8) 7.035 ,0.01 Stages III–IV 89 34 (38.2) 55 (61.8) cell (Figure 4B). Lymph node metastasis No 28 19 (67.9) 9 (32.1) 8.818 ,0.01 Yes 79 28 (35.4) 51 (64.6) MTA2 depletion inhibits growth, Distant metastasis metastasis, and Akt activity of CNE2 cell No 94 43 (45.7) 51 (54.3) 1.040 0.308 Yes 13 4 (30.8) 9 (69.2) in vivo We further explored the influence of MTA2 knockdown on Abbreviations: MTA2, metastasis-associated gene 2; NPC, nasopharyngeal carcinoma. growth and metastasis of CNE2 cell in vivo. Both control and CNE2/shMTA2 groups formed tumors in nude mice CNE1 and CNE2 cells were chosen for the gain-of-function after injection of tumor cells subcutaneously, but the tumor and loss-of-function studies, respectively. Stable MTA2- weight in CNE2/shMTA2 group was only 49% of the control overexpressing CNE1 cell (CNE1/MTA2) and negative group (Figure 5A). The tumor volume was also smaller in control (vector) were established by plasmid transfection. CNE2/shMTA2 group (data not shown). Furthermore, CNE2/ For personal use only. Stable MTA2-silencing CNE2 cell (shMTA2) and negative shMTA2 group formed much fewer pulmonary metastatic control (shNC) were established by lentivirus-mediated foci compared with control group after injection of tumor shRNA. cells through tail vein (Figure 5B). Finally, the expression of We found a 25-fold increase in MTA2 mRNA level and MTA2, pAkt, cyclin D1, and MMP7 proteins in subcutaneous a remarkable upregulation in MTA2 protein level in CNE1/ tumors of CNE2/shMTA2 group were lower than the control MTA2 cell compared with control (Figure 2A). CNE1/ group (Figure 5C). MTA2 cell had a higher proliferation rate in CCK-8 assay and formed more colonies in colony formation assay as Discussion compared with control (Figure 2B and C). The influence Although MTA2 is reported to be overexpressed in several of MTA2 upregulation on migration and invasion of NPC human malignancies and involved in the progression of OncoTargets and Therapy downloaded from https://www.dovepress.com/ by 54.70.40.11 on 13-Dec-2018 cell was investigated by wound healing assay and transwell tumor,13 little is known about the role of MTA2 in NPC. assay. Compared with control, CNE1/MTA2 cell had a higher For the first time, we identified the upregulation of MTA2 in wound closure rate (Figure 2D). Transwell invasion assay NPC tissues. Moreover, we also confirmed an overexpression also showed a significant increase in the number of invaded of MTA2 in three NPC cell lines detected compared with cells in CNE1/MTA2 cell (Figure 2E). nasopharyngeal epithelium cell line NP69. We concluded that MTA2 was upregulated in NPC and may participate in MTA2 depletion suppresses migration the carcinogenesis of NPC. and invasion of CNE2 cell in vitro In NPC tissues, MTA2 protein appeared in the nucleus MTA2 expression was successfully attenuated by lentivirus- of tumor cells and no obvious cytoplasmic staining was mediated shRNA determined by qRT-PCR and Western observed, which is consistent with previous studies.26 MTA2 blot (Figure 3A). MTA2 depletion inhibited proliferation of expression was positively related to clinical stage and lymph CNE2 cells by CCK-8 and colony formation assay (Figure 3B node metastasis of patients, indicating that MTA2 might be and C). MTA2 depletion also impaired the migration and indispensable in the progression of NPC. invasion ability of CNE2 cell, which was identified by wound In the three NPC cell lines detected, CNE2 cell had the healing and transwell assay (Figure 3D and E). highest and CNE1 cell had the lowest level of MTA2 protein.

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Figure 2 MTA2 overexpression promotes proliferation and migration of CNE1 cells. Notes: (A) MTA2 mRNA and protein levels of control and CNE1/MTA2 cells detected by qRT-PCR and Western blot. (B and C) Detection of cell viability by CCK-8 and colony formation assay. Detection of cell migration and invasion capability by wound healing ([D] magnification:× 100) and transwell invasion assay ([E] magnification:× 200); (*P,0.01). Abbreviations: MTA2, metastasis-associated gene 2; mRNA, messenger RNA; OD, optical density; qRT-PCR, quantitative real-time reverse transcription–polymerase chain reaction; CCK-8, Cell Counting Kit-8; h, hours.

In order to perform functional study, MTA2 was upregulated in CNE2 cell produced opposite effects. The xenograft in CNE1 cell and downregulated in CNE2 cell. We found experiments verified that MTA2 depletion attenuated cell that ectopic overexpression of MTA2 in CNE1 cell improved growth and metastasis in vivo. The results highlighted the cell viability and colony efficiency as well as cell migration importance of MTA2 in proliferation, migration, and inva- and invasion ability. On the other hand, MTA2 depletion sion of NPC cells.

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Dovepress MTA2 promotes proliferation and invasion of NPC cell

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OncoTargets and Therapy downloaded from https://www.dovepress.com/ by 54.70.40.11 on 13-Dec-2018 Figure 3 MTA2 knockdown inhibits proliferation and migration of CNE2 cells. Notes: (A) Analysis of MTA2 expression by qRT-PCR and Western blot in CNE2/shNC and CNE2/shMTA2 cells. (B and C) Analysis of cell viability and colony efficiency by CCK-8 and colony formation assay. (D and E) Analysis of cell migration and invasion ability by wound healing ([D] magnification: ×100) and transwell invasion assay ([E] magnification:× 200); (*P,0.01). Abbreviations: MTA2, metastasis-associated gene 2; OD, optical density; qRT-PCR, quantitative real-time reverse transcription–polymerase chain reaction; mRNA, messenger RNA; CCK-8, Cell Counting Kit-8; h, hours.

MTA2 had been reported to influence tumor cell growth The PI3K/Akt signaling pathway plays an important and migration. For example, ectopic expression of MTA2 role in cell proliferation, differentiation, and migration.31,32 promoted tumor cell growth and migration in gastric cancer Several studies indicated a close relationship between MTA1 and stimulated interleukin 11 secretion.27 In human breast (a member of MTA family with a structure similar to MTA2) cancer, MTA2 overexpression promoted cell invasion by and Akt signaling pathway.33,34 Moreover, silencing of MTA2 activating RhoA pathway.28 MTA2 depletion affected the changed the expression of some downstream genes of Akt expression of apoptosis-related genes P21, P27, P53, and pathway such as P21 and P53,15,29 suggesting a possible Bax and metastasis-related genes MMP2 and MMP9.29 interaction between MTA2 and Akt pathway. Therefore, MTA2 had been proved to influence cell apoptosis.30 The we compared the protein expression of Akt, pAkt, and some impact of MTA2 on apoptosis of NPC cell is worthy of downstream genes such as cyclin D1 and MMP7 in NPC cells. further investigation. Akt was activated by MTA2 upregulation and suppressed

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Figure 4 MTA2 stimulates Akt signaling pathway in NPC cells. Notes: Western blot analysis of Akt, pAkt, HIF-1α, MMP7, and cyclin D1 proteins expression in CNE1/vector and CNE1/MTA2 cells (A) or CNE1/shNC and CNE2/shMTA2 cells (B). Abbreviations: MTA2, metastasis-associated gene 2; NPC, nasopharyngeal carcinoma; HIF-1α, hypoxia-inducible factor 1α; MMP7, matrix metalloproteinase 7.

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Figure 5 Knockdown of MTA2 suppresses growth and metastasis of CNE2 cells in vivo. Notes: (A) Tumor size and tumor weight of CNE2/shNC and CNE2/shMTA2 groups in nude mice 3 weeks after injection of control or CNE2/shMTA2 cells subcutaneously in the right subaxillary. (B) Observation of pulmonary metastasis foci (black arrow) of nude mice 6 weeks after injection of control or CNE2/shMTA2 cells through tail vein (H&E ×100, *P,0.01). (C) Levels of MTA2, pAkt, cyclin D1, and MMP7 proteins in subcutaneous tumors of CNE2/shNC and CNE2/shMTA2 groups. Abbreviations: MTA2, metastasis-associated gene 2; H&E, hematoxylin and eosin; MMP7, matrix metalloproteinase 7.

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Dovepress MTA2 promotes proliferation and invasion of NPC cell

by MTA2 depletion, as well as protein levels of MMP7 and 10. Zhang B, Zhang H, Shen G. Metastasis-associated protein 2 (MTA2) cyclin D1. Nude mice experiment further verifies that MTA2 promotes the metastasis of non-small-cell lung cancer through the inhibition of the cell adhesion molecule Ep-CAM and E-cadherin. depletion inhibits Akt activity in vivo, indicating that MTA2 Jpn J Clin Oncol. 2015;45(8):755–766. might regulate expression of MMP7 and cyclin D1 via Akt 11. Liu YP, Shan BE, Wang XL, Ma L. Correlation between MTA2 overexpression and tumour progression in esophageal squamous cell pathway in NPC cells. carcinoma. Exp Ther Med. 2012;3(4):745–749. 12. Chen DW, Fan YF, Li J, Jiang XX. MTA2 expression is a novel prognostic marker for pancreatic ductal adenocarcinoma. Tumour Biol. 2013; Conclusion 34(3):1553–1557. In summary, we find that MTA2 is overexpressed in NPC 13. Covington KR, Fuqua SA. Role of MTA2 in human cancer. Cancer Metastasis Rev. 2014;33(4):921–928. tissues and cell lines. Upregulation of MTA2 promotes pro- 14. Kumar R, Wang RA, Mazumdar A, et al. A naturally occurring MTA1 liferation and invasion of NPC cells, while downregulation variant sequesters oestrogen receptor-alpha in the cytoplasm. Nature. 2002;418(6898):654–657. of MTA2 impairs cell growth and invasion, both in vitro and 15. Cheng CY, Chou YE, Ko CP, et al. Metastasis tumor-associated protein-2 in vivo. Furthermore, MTA2 silencing inhibits Akt pathway. knockdown suppresses the proliferation and invasion of human glioma This work strongly suggests that MTA2 plays an important cells in vitro and in vivo. J Neurooncol. 2014;120(2):273–281. 16. Zhou C, Ji J, Cai Q, et al. MTA2 promotes gastric cancer cells invasion role in the development of NPC and may be a potential and is transcriptionally regulated by Sp1. Mol Cancer. 2013;12:102. therapy target. 17. Fu J, Qin L, He T, et al. The TWIST/Mi2/NuRD protein complex and its essential role in cancer metastasis. Cell Res. 2011;21(2):275–289. 18. Soumaoro LT, Uetake H, Higuchi T, et al. Cyclooxygenase-2 expression: a significant prognostic indicator for patients with colorectal Acknowledgments cancer. Clin Cancer Res. 2004;10(24):8465–8471. This work is supported by the National Natural Science 19. Tsao SW, Wang X, Liu Y, et al. Establishment of two immortalized Foundation of China (81171824, 81371719, and 81371509), nasopharyngeal epithelial cell lines using SV40 large T and HPV16E6/E7 viral oncogenes. Biochim Biophys Acta. 2002;1590(1–3):150–158. Colleges Pearl River Scholar Funded Scheme (GDUPS, 20. Ma L, Deng X, Wu M, Zhang G, Huang J. Down-regulation of 2013), the Foundation for Distinguished Young Teachers Plan miRNA-204 by LMP-1 enhances CDC42 activity and facilitates inva- in Higher Education of Guangdong Province (YQ2013087), sion of EBV-associated nasopharyngeal carcinoma cells. FEBS Lett.

For personal use only. 2014;588(9):1562–1570. and the Research foundation of Guangdong Medical 21. Livak KJ, Schmittgen TD. Analysis of relative gene expression data University (M2012002, M2015011). using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402–408. 22. Deng X, Ma L, Wu M, et al. miR-124 radiosensitizes human glioma cells by targeting CDK4. J Neurooncol. 2013;114(3):263–274. Disclosure 23. Shen Z, Zeng Y, Guo J, et al. Over-expression of the special AT rich The authors report no conflicts of interest in this work. sequence binding protein 1 (SATB1) promotes the progression of nasopharyngeal carcinoma: association with EBV LMP-1 expression. J Transl Med. 2013;11:217. References 24. Caysa H, Hoffmann S, Luetzkendorf J, et al. Monitoring of xenograft 1. Wei KR, Zheng RS, Zhang SW, Liang ZH, Ou ZX, Chen WQ. Nasopha- tumor growth and response to chemotherapy by non-invasive in vivo ryngeal carcinoma incidence and mortality in China in 2010. Chin J multispectral fluorescence imaging.PLoS One. 2012;7(10):e47927. Cancer. 2014;33(8):381–387. 25. Jiang H, Gao M, Shen Z, et al. Blocking PI3K/Akt signaling attenuates

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